Methods for freeze-drying bacteria and utility as a mucosal vaccine platform

Abstract

Embodiments of the present disclosure generally relate to compositions of a lyophilized (freeze dried) Listeria strain based therapeutics and a method for lyophilizing compositions of the therapeutics. A Listeria monocytogenes (LM) based vaccine formulation contains a lyophilized powder including a LM bacteria, the LM bacteria comprising at least one therapeutic agent and a lyophilization medium and a delivery composition. A method of forming a Listeria monocytogenes (LM) based vaccine formulation is disclosed. The LM based vaccine formulation includes resuspending an LM stock in a lyophilization medium to form a lyophilization composition, wherein the lyophilization medium comprises at least a lyoprotectant and a buffer component, freezing the lyophilization composition, lyophilizing the lyophilization composition, and forming a lyophilized powder, the lyophilized powder contains a LM bacteria, a lyoprotectant, and a buffer component.

Description

PATENTAttorney Docket No.: TXTU / 0006PCMETHODS FOR FREEZE-DRYING BACTERIA AND UTILITY AS A MUCOSAL VACCINE PLATFORMBACKGROUNDField

[0001] Embodiments of the present disclosure generally relate to compositions of a lyophilized (freeze dried) Listeria strain based therapeutic, method for lyophilizing compositions of the therapeutic, and utility as a mucosal vaccine platform.Description of the Related Art

[0002] Lyophilization is a commonly used method of preparing bacteria for long term storage or for everyday use. There is a major emphasis on identifying the best methods for preparation and storage to ensure high stability and function. In line with this, lyophilization is one of the major methods used to preserve stability, particularly for bacterial and viral-based vaccines (e.g., Listeria strain-based vaccines). The lyoprotectant and buffer components of a lyophilization medium greatly impact bacterial viability.

[0003] Listeria monocytogenes (LM) is a Listeria strain. LM is a Gram-positive intracellular bacterium currently being investigated preclinically and clinically as an anticancer vaccine platform. LM based tumor vaccines utilize attenuated strains to elicit high antitumor activity. The ability of LM based vaccines to enter the cellular environment, overcome immunosuppressive cells, and trigger targeted destruction of cancer cells by activating cytotoxic T cells has been demonstrated. LM based vaccines are being explored for numerous different tumor applications and have shown success including cervical, prostate, esophageal, pancreatic, rectal, colorectal, and lung clinically and many others preclinically. However, LM based tumor vaccines typically are stored at ultra-low temperatures (ULT). ULT storage requires equipment capable of maintaining temperatures in the range of -20°C to -80°C. Numerous challenges to ULT logistics include stringent operating conditions, high energy demands, the need for reliable power supply, and transportation. Higher cold chain storage temperatures such as chilled (2°C to 4°C) and frozen (-16°C to -20°C) are more widely available and include fewer challenges when compared to ULT logistics.PATENTAttorney Docket No.: TXTU / 0006PC

[0004] Mucosal-derived cancers are among the deadliest. Mucosal delivery of a therapeutic vaccine would stimulate anti-tumor immunity which preferentially targets mucosal tissues, likely providing greater anti-cancer efficacy. LM’s natural life cycle as a foodborne pathogen makes it well-suited for application as a mucosal vaccine platform. However, LM-based vaccines are typically administered systemically, likely limiting efficacy against mucosal-derived malignancies.

[0005] Therefore, there is a need for a composition of a LM based therapeutic including a lyoprotectant and buffer components as well as a method of lyophilizing the LM, lyoprotectant, and buffer components to allow for stability during storage and delivery to mucosal sites.SUMMARY

[0006] In one embodiment, a lyophilized composition is provided and contains a Listeria monocytogenes (LM) bacteria and a lyophilization medium. The lyophilization medium including a lyoprotectant and a buffer component.

[0007] In another embodiment, a Listeria monocytogenes (LM) based vaccine formulation is disclosed. The LM based vaccine formulation contains a lyophilized powder including a LM bacteria, the LM bacteria comprising at least one therapeutic agent and a lyophilization medium and a delivery composition.

[0008] In another embodiment, a method of forming a Listeria monocytogenes (LM) based vaccine formulation is disclosed. The LM based vaccine formulation includes resuspending an LM stock in a lyophilization medium to form a lyophilization composition, wherein the lyophilization medium comprises at least a lyoprotectant and a buffer component, freezing the lyophilization composition, lyophilizing the lyophilization composition, and forming a lyophilized powder, the lyophilized powder contains a LM bacteria, a lyoprotectant, and a buffer component.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appendedPATENTAttorney Docket No.: TXTU / 0006PC drawings illustrate only exemplary embodiments of the disclosure and are therefore not to be considered limiting of the scope of the disclosure, and may admit to other equally effective embodiments.

[0010] Figures 1 A, 1 B, 1 C, 1 D, and 1 E provide a comparison of LM lyophilization media under freezer and refrigeration storage conditions over a course of one month using different formulations and storage temperatures, according to certain embodiments.

[0011] Figure 2 shows a comparison of reported commercial microbial lyophilization mediums, according to certain embodiments.

[0012] Figure 3 shows examples of lyophilized LM based therapeutics, according to certain embodiments

[0013] Figure 4 shows a long term stability of LM in lyophilization mediums, according to certain embodiments.

[0014] Figures 5A, 5B, 5C, and 5D show the characterization of buffer components with or without LM, according to certain embodiments.

[0015] Figure 6 shows a residual moisture content of lyophilized LM, according to certain embodiments.

[0016] Figure 7 shows an in vivo comparison of lyophilized antivascular LM LLO- CD105A vaccine in a subcutaneous RCC model, according to certain embodiments.

[0017] Figure 8 shows immune infiltration of (A) leukocytes (CD45+) and (B) MDSC (CD11 b+GR1hi) in RENCA tumor cell environment post treatment with PBS, nonlyophilized stock LM LLO-CD105A, and lyophilized LM LLO-CD105A analyzed by flow cytometry, according to certain embodiments.

[0018] Figure 9 shows sulforhodamine B (SRB) release from coated M size capsules under (A) simulated intestinal fluid and (B and C) simulated gastric fluid over time. SRB capsule payloads are released rapidly under intestinal conditions with methacrylic acid copolymer (e.g., Eudragit S-100) and Polycaprolactone (PCL)PATENTAttorney Docket No.: TXTU / 0006PC prolonging release under gastric conditions thereby enabling intestinal targeted delivery, according to certain embodiments.

[0019] Figure 10 shows a visualization of M size capsules uncoated and coated with (A) methacrylic acid copolymer (e.g., Eudragit S-100) and (B) polycaprolactone (PCL) release of SRB dye under simulated gastric and intestinal conditions, according to certain embodiments.

[0020] Figure 1 1 shows a timeline representation and a quantification of tumor volume in an in v / vo treatment of orthotopic mammary fat pad implanted EO771 breast cancer with LM LLO-CD105A antivascular vaccine strain, according to certain embodiments.

[0021] Figure 12 is a flow diagram of a method of preparing LM for lyophilization, according to some embodiments.

[0022] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.DETAILED DESCRIPTION

[0023] Embodiments of the present disclosure generally relate to compositions of a lyophilized (freeze dried) Listeria strain based therapeutics, a method for lyophilizing compositions of the therapeutics, and application as a mucosal therapeutic. As disclosed herein, the Listeria strain is a recombinant Listeria monocytogenes (LM). However, it should be understood that other strains of Listeria may be used in the compositions disclosed herein or processed with the methods disclosed herein.

[0024] Lyophilization is a commonly used method of preparing bacteria for long term storage or for everyday use. The lyoprotectant and buffer components of a lyophilization medium greatly impacts bacterial viability and stability. LM is a Grampositive intracellular bacterium currently being investigated preclinically and clinically as an anticancer vaccine platform. LM has been applied therapeutically as an anticancer vaccine platform wherein encoded tumor antigens are secreted by LMPATENTAttorney Docket No.: TXTU / 0006PC inside target cells and promote a targeted adaptive immune response. In clinical and preclinical settings, LM anticancer vaccines demonstrate efficacy against primary and metastatic cancer. Additionally, conventional LM anticancer vaccines are inexpensive to produce once a therapeutic strain has been developed, and, therefore, have the potential to be broadly accessible form of tumor immunotherapy. The natural replication capabilities of LM, and other bacteria, can be grown in a laboratory culture media at scale, which allows for the accessibility.

[0025] Current formulations require storage at ultra-low temperatures, which severely limits the accessibility of LM therapies because many parts of the world lack the necessary equipment. For example, following preparation in conventional methods, aliquots of LM are stored at -80°C. Storage at -80°C is categorized as under ultra-low temperature (ULT) freezing, which causes logistical challenges in distribution and storage. ULT storage requires equipment capable of maintaining temperatures in a range of -60°C to -80°C. The World Health Organization (WHO) identifies numerous key challenges to ULT logistics including stringent operating conditions, high energy demands, the need for reliable power supply, and transportation. Access to the necessary equipment and logistics for ULT storage and / or transportation are not available in many areas of the word, which makes ULT dependent therapies inaccessible to parts of the world. Higher cold chain storage temperatures (e.g., chilled (2°C to 4°C) or frozen (-16°C to -20°C) allow for accessibility for more parts of the world. Accordingly, an LM anticancer vaccine operable to be stored at a higher temperature would increase availability worldwide.

[0026] Lyophilization of LM maintains high cellular viability and therapeutic potency of LM anticancer vaccines. Further, lyophilization (freeze-drying) allows for bacteria storage at chilled or frozen temperatures. Lyophilization is a process involving dehydrating a frozen sample using a strong vacuum that induces the sublimation of water (within the frozen sample) from a solid to a vapor. A sample may be frozen with a lyophilization media, which may include water, a lyoprotectant, and a buffer component. During freezing and lyophilization, a lyoprotectant serves to protect macromolecule stability during the removal of water. Common lyo protectants are sucrose, trehalose, and skim milk which all improve stability by forming hydrogen bonds with proteins within a sample to help maintain a natural structure during thePATENTAttorney Docket No.: TXTU / 0006PC lyophilization process. Transition glass temperature (Tg), the temperature at which an amorphous material transitions to a more mobile state, is a parameter measured during the lyophilization of a sample. In general, materials undergoing lyophilization should be kept below their respective Tg to enable water removal from the frozen material. Further, when bacteria are lyophilized, if the difference between the Tg and the storage temperature becomes too great there is a loss of storage stability. Thus, Tg is a predictive parameter for storage temperatures of lyophilized bacteria after drying. Typically, during freezing, solutions change from a homogenous liquid to heterogenous solids where solubilized components become concentrated. The concentration of the solubilized components can result in changes in pH during freezing and lyophilization, which can destabilize proteins. Lyophilization buffers are used during freezing and lyophilization to maintain bacterial stability. Lyophilization buffers are non-volatile, so that the lyophilization buffers are not removed by the vacuum during the lyophilization process.

[0027] Conventional lyophilization methods can include a variety of media components such as buffers, lyoprotectants, surfactants, viscosity reducers, and / or bulking agents to support preparation. However, many of the media components used in conventional lyophilization methods are detrimental to bacterial viability. For example, surfactants and viscosity reducers can disrupt the bacterial membranes and reduce cell viability, which hingers the ability of lyoprotectants to replace the hydrated layer of the cell membrane and cell wall to promote stability.

[0028] Accordingly, a lyophilization composition and a lyophilization method related to a Listeria strain is disclosed. Specifically a composition and method related to recombinant Listeria monocytogenes (LM) is disclosed. Further, an encapsulation composition and method is disclosed, where the lyophilized LM is encapsulated for administration to a subject.Materials and MethodsMice

[0029] All mice used in animal experiments were procured from Jackson Laboratories at 5-weeks old and were housed at the animal core facility at Texas TechPATENTAttorney Docket No.: TXTU / 0006PCUniversity Health Science Center (TTUHSC) Laboratory Animal Research Center (LARC) under BSL2 conditions. Animal care and experiments were performed in accordance with regulations provided by Institutional Animal Care and Use Committee of TTUHSC.Listeria Cell Culture

[0030] LM is cultured in brain heart infusion (BHI) media optionally supplemented with 34 pg / mL streptomycin (Thermo Scientific cat# 455340050) and chloramphenicol (ACROS Organics cat# 227920250), depending on encoded antibiotic resistances, at 37°C to an OD600 value between 0.65-0.75. In one or more embodiments, the LM stocks are cultured in a cell culture medium. LM stocks are stored at -80°C for no longer than 2 months. LM stock density is measured using CFU counting by serial dilution and streaking on BHI (BD Difco cat# 237500) agar plates (density between 1- 2x109CFU / mL). The LM LLO-Ova strain is developed from the XFL-7 (Ap / fA) strain using the pGG34 plasmid, which encodes a truncated Listeriolysin (LLO) fused to chicken ovalbumin. Similarly, the LM LLO-CD105A strain is developed using the pGG34 plasmid encoding the LLO-CD105A fusion antigen. In both plasmids, the encoded LLO fusion protein is expressed under the control of a prfA promoter and encodes a modified prfA transcription factor enabling partial rescue of XFL-7 attenuation.Cancer Cell Culture

[0031] The macrophage-like reticulum cell sarcoma cell line J774 and kidney cancer cell line Renca are used. All cells are maintained by culturing at 37 °C 5% CO2 in Dulbecco Modified Eagles Medium (DM EM) (Corning cat# 10-013-CV) supplemented with 10% FBS (Corning cat# 35-011-CV), 4.5 g / L glucose, L- glutamine, sodium pyruvate, 10 U / mL penicillin, 10 pg / mL streptomycin (Coming cat# 30-002-CI), and 5 pg / mL plasmocin (InvivoGen cat# ant-mpp). Cells were tested for mycoplasma contamination to verify there was no impact on experimentation. Subculturing of cells is performed using 0.25% trypsin (Corning cat# 25-053-CI) supplemented with 0.54 mM EDTA and 1X sodium bicarbonate and scraping in the case of J774.PATENTAttorney Docket No.: TXTU / 0006PC

[0032] Figure 12 is a flow diagram of a method 1200 of preparing LM for lyophilization.

[0033] At operation 1202, LM stocks are prepared. In one embodiment, a liquid LM culture is inoculated from an agar colony. The LM is grown on a shaker. The LM is grown in the shaker until the LM reaches an optical density (OD). The OD is about 0.65 to about 0.75. After the OD is achieved, the LM is aliquoted. In one or more embodiments, other means of preparing bacteria stock may be used at operation 1202. After preparation of the LM stocks, the LM stocks are aliquoted into individual vials (e.g., Eppendorf tubes or Falcon tubes). The aliquoted LM stocks are frozen. In one example, the aliquoted LM stocks are frozen at about -40°C to about -90°C. The samples are held in the freezer. The samples may be held in a freezer for at least three hours. Colony forming unit (CFU) testing is performed on the aliquoted LM stocks to determine an initial CFU of the samples. The CFU data is used to calculate efficiency of the lyophilization process. In one or more embodiments, each stock includes about 1 billion CFU / mL to about 1.7 billion CFU / mL.

[0034] The LM includes at least one therapeutic agent. For example, the LM may be associated with one or more therapeutic agents. The therapeutic agents may be associated with the surface of the LM through a binding agent. For example, the binding agent is simultaneously associated with the surface of the LM and the therapeutic agent. The binding agent is associated with the therapeutic agent via a cleavable linker through non-covalent bonding. Alternatively or additionally, the therapeutic agents may be associated with the surface of the LM through a linker. The linker directly links the therapeutic agent to the surface of the LM. For example, the therapeutic agents may include saporin, an active metabolite of CPT-11 (e.g., SN38), doxorubicin, derivatives thereof, or combinations thereof. The LM with at least one therapeutic agent may be suitable for use in treating a cancer (e.g., ovarian cancer, colorectal cancer, sarcoma, or hepatocellular carcinoma). Additionally or alternatively, the therapeutic may be expressed and produced by the LM. The therapeutic may include, which may or may not be secreted by the LM, anticancer vaccine antigens, cytotoxins, and / or cell modulating signals which promote anticancer activity. LM based vaccine formulations or therapeutics can infect cells, secrete tumor targeting antigens, and stimulate a directed immune response against tumors forPATENTAttorney Docket No.: TXTU / 0006PC therapeutic effect. For example, the LM LLO-CD105A vaccine primarily stimulates an adaptive immune response where CD8+T cells are the primary antitumoral effectors. Successful stimulation following LM injection requires functional proteins and other biomolecules which must be preserved during the lyophilization process. Successful immune stimulation requires a minimal recovery time following rehydration of the LM. The equivalent efficacy of LM LLO-CD105A, therefore, demonstrates that the lyophilized LM are well preserved and quickly active following the rehydration of the LM. The injection of non-viable LM in treatments plays a role in the increased immune recruitment to tumor sites. Myeloid derived suppressor cell’s (MDSC) are highly immunosuppressive and pro-tumoral and an increased recruitment to tumor sites by lyophilized LM treatment could be synergistically targeted with immune checkpoint therapy. Preservation of antitumor efficacy but an increase in immune recruitment to tumor sites by treatment with lyophilized LM vaccines could indicate there are advantages to using lyophilized LM over storage of liquid stocks at ULT.

[0035] At operation 1204, the LM stocks are thawed. In one embodiment, the aliquoted individual vials are removed from the freezer. The aliquoted individual vials are held outside of the freezer at a temperature until the LM stock is thawed completely. For example, the aliquoted LM stocks are held in a water bath. The temperature the aliquoted LM stocks are held at is about 30°C to about 40 °C. The LM stocks are centrifuged. The LM stocks are centrifuged at a relative centrifugal force (RCF) of about 4,000 g-force (xg) to about 8,000 xg. The LM stocks are centrifuged for about 5 min to about 10 min. In one example, the LM stocks are centrifuged at about 4,000 xg to about 8,000 xg for about 5 min to about 10 min. Operation 1204 results in pelleted LM cells and a cell culture medium supernatant.

[0036] At operation 1206, the cell culture medium supernatant of the LM stock is removed from each aliquot of the LM stock. The LM stock (e.g., the LM cells that were pelleted during operation 1204) is resuspended in a lyophilization medium forming a lyophilization composition. The lyophilization medium may also be referred to as a lyophilization buffer. The lyophilization medium includes components that produce a lyoprotectant and a buffer component after lyophilization. For example, each LM stock, which includes about 1 billion CFU to about 1.7 billion CFU, is resuspended in about 400 pL to about 600 pL of lyophilization medium. The amountPATENTAttorney Docket No.: TXTU / 0006PC of lyophilization medium used for resuspension is determined by the ratio of bacteria (e.g., the LM) per lyoprotectant and buffer components. For example, the ratio of lyophilization medium can be 300 pL to 500 L in volume per 1 billion CFU to about 1.7 billion CFU resuspended. In one or more embodiments, the amount of lyophilization medium used for resuspension is determined by the CFU density. For example, the CFU density may be between about 1x109CFU to about 1x1010CFU. For example, the expected lyophilized density is about 2x109CFU / mL to about 2x1010CFU / mL. The density of about 1x109CFU to about 1x1010CFU results in an expected lyophilized density of about of 2x109CFU / mL to about 2x1010CFU / mL. Generally, for example, the CFU density may be about 1x109to about 3x1010CFU / mL.

[0037] The lyophilization medium may be any lyophilization media example disclosed in T able 1 . Further embodiments of the lyophilization medium are described in Paragraph

[0038] , Table 1 discloses each lyophilization medium tested and the constituents of each lyophilization medium. In one or more embodiments, each lyophilization medium listed in Table 1 is prepared in 18.2 mQ / cm milli-Q (MQ) water with buffer constituents at concentrations described in Table 1. The buffer constituents include at least the lyoprotectant and the buffer component. The pH of lyophilization media of Table 1 is adjusted by addition of sodium hydroxide and / or hydrochloric acid solution until the pH is about 6 to about 8. For example, the pH of the lyophilization media is adjusted to a pH of about 7.4. All media are sterilized and stored under refrigeration conditions.

[0038] In one or more embodiments, the lyophilization medium includes a 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) sucrose medium. The HEPES sucrose medium includes about 1 millimolar (mM) to about 10 mM of HEPES and about 2% to about 10% weight / volume (w / v) of sucrose. Forexample, the HEPES sucrose medium includes about 3 mM of HEPES and about 2.5% w / v of sucrose. In one or more embodiments, the lyophilization medium includes a triethanolamine (TEA)sucrose medium. The TEA sucrose medium includes about 10mM to about 40 mM of TEA HCI and about 2% to about 10% w / v of sucrose. For example, the TEA sucrose medium includes about 2.5% w / v of sucrose. In one or more embodiments, the lyophilization medium includes a HEPES cellulose medium. For example, the HEPES cellulose medium includes 1 mM to about 10 mM of HEPES and about 2.5%PATENTAttorney Docket No.: TXTU / 0006PC w / v of sucrose. In one or more embodiments, the lyophilization medium includes a glutamate sucrose medium. The glutamate sucrose medium includes about 50 mM to about 100 mM of sodium glutamate and about 2% to about 10% w / v of sucrose. In one or more embodiments, the lyophilization medium includes a HEPES phosphate sucrose medium. The HEPES phosphate sucrose medium includes about 1 mM to about 10 mM of HEPES, about 1 mM to about 3 mM of potassium phosphate, about 5 mM to about 10 mM of sodium phosphate, and about 2% to about 10% w / v of sucrose. For example, the HEPES phosphate sucrose medium includes about 2.5% w / v of sucrose. The HEPES cellulose medium includes about 1 to about 10 mM of HEPES and about 2% to about 10% w / v of cellulose. For example, the HEPES cellulose medium includes about 2.5% w / v of cellulose. In one or more embodiments, the lyophilization medium includes a HEPES trehalose medium. The HEPES trehalose medium includes about 1 mM to about 10 mM of HEPES and about 2% to about 10% w / v of trehalose. For example, the HEPES trehalose medium includes about 2.5% w / v of trehalose. In one or more embodiments, the lyophilization medium includes a HEPES sucralose medium. The HEPES sucralose medium includes about 1 mM to about 10 mM of HEPES and about 2% to about 10% w / v of sucralose. For example, the HEPES sucralose medium includes about 2.5% w / v of sucralose. In one or more embodiments, the lyophilization medium includes a HEPES xylitol medium. The HEPES xylitol medium includes about 1 mM to about 10 mM of HEPES and about 2% to about 10% w / v of xylitol. For example, the HEPES xylitol medium includes about 2.5% w / v of xylitol.Table 1PATENTAttorney Docket No.: TXTU / 0006PC

[0039] At operation 1208, the lyophilization composition (e.g., the LM stock and the lyophilization medium) is aliquoted into ampules. The ampules are frozen after the lyophilization composition is aliquoted into ampules. The temperature for freezing the ampules is -40°C to about -90°C. The ampules remain in the freezer until the lyophilization composition is completely frozen. The time for the lyophilization composition to completely freeze is at least 30 minutes. In one example, the ampules remain in the -80°C freezer for at least 30 minutes and up to 3 hours.

[0040] At operation 1210, the lyophilization composition is lyophilized. The ampules containing the lyophilization composition are placed on a means of freeze drying. The means forfreeze drying is a lyophilizer. The lyophilizer includes a vacuum operable to maintain a pressure of about 0.1 mbar to about 0.9 mbar. The temperature of the lyophilizer is selected to condense water from the ampules and prevent it from entering the vacuum. In one embodiment, the temperature is about - 30 to about -80°C. The lyophilization composition ampules containing the lyophilization composition are retained in the means for freeze drying for about 14 hours to about 18 hours. When a lyophilizer is used, the ampules containing the lyophilization composition remain on the vacuum until completely freeze-dried (e.g., there is no liquid or frozen portions of the lyophilization composition).

[0041] In one or more embodiments, after ensuring resuspension, the LM in the lyophilization medium within an ampule (e.g., an amber glass ampule), each ampule is transferred and sealed within a lyophilization chamber and frozen at -80°C for about 3 hours. Once the lyophilization composition is frozen within each ampule, the lyophilization chamber is coupled to a lyophilizer (e.g., a benchtop free dry system) and chilled to about -50°C. In an example, the lyophilization composition is lyophilizedPATENTAttorney Docket No.: TXTU / 0006PC under vacuum between about 0.25 mbar to about 0.75 mbar overnight. For example, the lyophilization composition is lyophilized for about 16 hours to about 18 hours (e.g., overnight).

[0042] At operation 1212, the ampules are removed from the lyophilizer and sealed. For example, the ampules may be sealed with an acetylene torch. At operation 1212, the ampules include a lyophilized powder. The lyophilized powder is a lyophilized lyophilization composition. The lyophilized powder includes the LM, the lyoprotectant, and the buffer components.

[0043] At operation 1214, the ampules of the lyophilized powder are stored. The ampules of the lyophilized powder are stored at 2°C to 4°C or -16°C to -20°C. In one or more embodiments the lyoprotectant is sucrose, trehalose, sucralose, xylitol, cellulose or combinations thereof. In one or more embodiments, the buffer component is HEPES, glutamate, TEA or combinations thereof. In one or more embodiments, the buffer component further includes a phosphate component (e.g., derived from a phosphate buffer). In one or more embodiments, the lyophilized powder is operable to be stored without a negative impact at 2°C to 4°C for about 1 day to about 1 month. In one or more embodiments, the lyophilized powder is operable to be stored without a negative impact at or -16°C to -20°C for about 1 day to about 3 months.

[0044] After the method 1200, the lyophilized powder is removed from storage. The lyophilized powder is removed from storage to test survival percentage of the LM after storage at 2°C to 4°C or -16°C to -20°C. The lyophilized powder is resuspended in phosphate buffered saline (PBS). For example, the volume of PBS used to reconstitute the lyophilized powder is at least about 100 pL. LM CFU density was determined using a CFU counting method. After determining the total CFU, the survival of LM following lyophilization and storage is determined using the equation: Survival% = (LyoCFU / -80°C StockCFU) x100. Survival measurements of lyophilized LM were taken at 1 day, 2 week, and 4 week increments to evaluate stability. In one or more embodiments, the lyophilized LM included a survival percentage of about 50% to about 115%, or about 75% to about 100%.

[0045] The resulting lyophilized powder produced by method 1200 is operable to be a therapeutic. For example, the lyophilized powder is a lyophilized LM LLO-PATENTAttorney Docket No.: TXTU / 0006PCCD105A treatment when LM LLO-CD105A is used as the LM strain during processing. Further processing of the lyophilized powder that contains the LM, the lyoprotectant, and buffer components may occur. The LM based therapeutics and associated method described herein have maintained viability of the LM while maintaining the infectivity and antitumor efficacy of the LM. For example, as disclosed below the LM show antitumor efficacy in a subcutaneous renal cell carcinoma model. The lyophilization method described herein is adoptable for the preparation of LM and allows for stable storage at non-ultra-low temperatures.

[0046] The LM based therapeutic includes the lyophilized powder formed by the method 1200. The lyophilized powder includes the lyophilized LM, the buffer component, and a lyoprotectant. In one or more embodiments, a ratio of the lyophilized LM to the buffer component is about 200 million CFU per milligram of buffer to about 4 billion CFU per milligram of buffer. In one or more embodiments, a ratio of the lyophilized LM to the lyoprotectant is about 10 million CFU per mg of lyoprotectant to about 250 million CFU per mg of lyoprotectant. The lyophilized powder may be used in LM based vaccine formulation or therapeutic. The lyophilized powder may be resuspended in a liquid for resuspension such as a PBS buffer, normal saline, or media for use in vaccine administration. The amount of suspension buffer is determined by the density of the surviving colonies of the LM in the lyophilized powder and the desired amount for injection in a desired volume. For example, for injection studies it is desirable to inject about 1 x 107CFU to 1 x 109CFU of the LM depending on method of injection and subject. Accordingly, any volume of resuspension liquid may be added to the lyophilized powder to reach the desired density.

[0047] In one or more embodiments, the lyophilized powder produced by method 1200 is further processed such that the LM based vaccine formulation may be administered intravenously, intraperitoneally, or orally. In one or more embodiments, the LM based vaccine formulation includes a delivery composition. The delivery composition includes the means to deliver at least the therapeutic agent to a subject. For example, the delivery composition includes a liquid for resuspension, a capsule, or any other means for delivery to the subject. The disclosed LM based vaccine is a mucosal vaccine platform. The vaccine is delivered through mucosal surfaces of thePATENTAttorney Docket No.: TXTU / 0006PC subject such as the nose, mouth, or gastrointestinal tract. Using a mucosal vaccine platform to deliver the disclosed LM based vaccine formulation allows for a targeted delivery of the therapeutic agent (e.g., a targeted delivery instead of a systematic delivery). The targeted delivery allows for a direct immune activation, an enhanced cellular response, promotes a local immunity, and reduces side effects associated with the systematic delivery.

[0048] In one or more embodiments, the lyophilized powder may be resuspended in a liquid as described above to include a particular CFU and injected into a subject. In one or more embodiments, the lyophilized powder is further processed with a capsule preparation protocol. In one or more embodiments, the lyophilized powder is encapsulated for use in oral administration. In one or more embodiments, the lyophilized powder is encapsulated in an acid resistant capsule. In one or more embodiments, the capsule may further include a coating such as acrylate polymers, hydroxypropyl methylcellulose phthalate, and polyvinyl acetate phthalate such that passage through the stomach and release in the intestines is ensured. It is contemplated that encapsulation allows for a controlled release of the therapeutic in the mid intestine, lower intestine, or colon regions of the Gl tract. This enables a greater therapeutic value and efficacy of the administered LM.

[0049] In one or more embodiments, first, a capsule holder, funnel, and open capsule are prepared. The capsule may be any available capsule of a size appropriate to contain an appropriate amount of the lyophilized powder. For example, the size of the capsule is dependent on the type of subject or the size of the subject. Next, about 100 million CFU to about 500 million CFU of the lyophilized powder is divided out. For example, about 200 million CFU are separated in preparation for insertion into an M sized capsule. However, it should be understood that any amount of lyophilized powder that provides an appropriate amount of therapeutic may be separated and inserted in each capsule. For example, the volume of the lyophilized powder may contain approximately one billion CFU to approximately 3 billion CFU of LM per capsule for use by human subjects. However, it should be understood that any amount of CFU to be an effective therapeutic is contemplated. The lyophilized powder is placed through the funnel and compressed into the open capsule. The capsule is then closed and checked for deformation.PATENTAttorney Docket No.: TXTU / 0006PC

[0050] Each capsule is then coated with a protective coating. For example, the coating is methacrylic acid copolymer, polycaprolactone (PCL), cellulose derivatives, polyvinyl derivatives, or combinations thereof. The methacrylic acid copolymer is operable to dissolve at high pH of about 6 or higher such that the coated therapeutic dissolves in the intestine rather than the stomach. PCL is a biodegradable and biocompatible polyester that is operable to provide a controlled release of a therapeutic. For example, the methacrylic acid copolymer coating includes about 5- 10% w / v methacrylic acid copolymer, 0.5-1 % w / v triethyl citrate, 2-4% w / v magnesium stearate, 30-45% volume of acetone, 50-60% volume isopropanol, and 5-10% water. The PCL includes about 70 to about 90 kilodalton (KDa) PCL at 5-10% w / v and dichloromethane. Each capsule may be coated 1 time to 3 times to ensure a uniform coating. Each capsule is dried for about 5 minutes to about 15 minutes between coatings. After coating, the capsules can be stored until administration at non-ULT temperatures (e.g. , 2°C to 4°C or -16°C to -20°C) for about 1 month to about 3 months.

[0051] The methods of preparing lyophilized LM described herein are readily applicable and do not require specialized equipment. Requiring only a freeze-drying manifold and vacuum capable of achieving a pressure of about 0.2 mbar to about 0.8 mbar, the ease of lyophilized LM preparation makes adoption of such protocols a simple process. Furthermore, as demonstrated here, lyophilized LM can be stored at non-ULT temperatures (e.g., 2°C to 4°C or -16°C to -20°C), which greatly increases the accessibility of LM anticancer vaccines globally.Testing MethodsListeria Infectivity Gentamicin Protection Assay

[0052] For comparison of LM infectivity, a gentamicin protection assay was developed and adapted from conventional protocols. J774 cells were passaged as normal and seeded into a 96-well tissue culture plate at a density of 32,500 cells / well. After about 24 hours, in preparation for infecting cells, -80°C LM stocks were thawed rapidly in a 37°C water bath or were resuspended with 1 mL of PBS in the case of lyophilized LM. LM were centrifuged at 5,000xg for 10 min and were resuspended in room temperature cell culture media supplemented with 10% fetal bovine serum (FBS) with no anti-biotics and diluted to desired multiplicity of infection (MOI)PATENTAttorney Docket No.: TXTU / 0006PC densities. About 22 hours to about 24 hours post cell seeding, J774 cells were washed once with PBS media and 100 pL of LM containing media was added to each well. The cells were then placed into a culture incubator and infected for about 3 hours. Following infection, the media was removed and the cells were washed once with room temperature PBS. 100 pL of fresh media is added and the cells are left at room temperature for about 15 minutes for a short recovery period follows by another 100 pL of media containing 100 pg / mL gentamicin (ACROS organics cat# 1405-41-0) to achieve a final gentamicin concentration of about 50 pg / mL. Following another incubation period of about 1 hour at about 37°C, the media is removed and replaced with warm MQ water to lyse the cells. Following lysis, wells were thoroughly mixed by pipetting, serially diluted into PBS and the CFU titrated on brain heart infusion (BHI) agar plates (Fisher Scientific cat#FB0875711 A square dishes).Listeria Cytotoxicity Cell Viability Assay

[0053] For comparison of LM cytotoxicity, a gentamicin protection assay (GPA) assay is used in conjunction with a sulforhodamine B (SRB) cytotoxicity assay. J774 cells are seeded in a 96-well tissue culture plate at a density of 10,000 cells / well. After 24 hours, LM are prepared at desired densities in antibiotic lacking media. J7774 cells are washed once with PBS before the addition to the 96-well tissue culture plate. Cell infection is performed at 37°C for about 3 hours. Following infection, media is removed, the cells are washed with PBS, and 100 pL of antibiotic lacking media is added to the wells for a 15 minute recovery period. 100 pL of media containing 10 pg / mL gentamicin is added to achieve a final gentamicin concentration of 5 pg / mL. Infected cells are incubated for about 48 hours with fresh 5 pg / mL gentamicin media added after the first 24 hours. Following the incubation period, cells are fixed with 10% trichloroacetic acid (SAFC cat# T06999) overnight at 2°C to 4°C. Following four washes with MQ water the 96-well tissue culture plate is air dried and 100 pL of 0.4% SRB dye (TCI cat# A0600) in 1 % acetic acid (Fisher Chemical cat# A38A-212) was added to each well. Cells are stained for 30 minutes in the dark at room temperature. After staining, cells are washed 4 times with 1 % acetic acid solution. The 96-well tissue culture plate is air dried before adding 10 mM Tris base (Sigma Aldrich cat# 77- 86-1 ) and absorbance at 565 nm was measured to determine cytotoxicity.PATENTAttorney Docket No.: TXTU / 0006PCScanning Electron Microscopy (SEM) Imaging

[0054] Lyophilized samples were prepared as described above and sealed in ampoules until ready for SEM analysis. For preparation of SEM imaging all materials are handled in a biosafety hood. To mount lyophilized samples for analysis, the powders are lightly pulverized with a cleaned spatula and then stuck to the microscope mount by compression to an adhesive carbon tape. Samples are immediately imaged using a JEOL JSM-IT200 scanning electron microscope located at Abilene Christian University. Images are taken in the normal scanning electron detector (SED) imaging mode at high vacuum conditions with an accelerating voltage of 20 kV, a probe current set to 30, and a working distance of 15 mm.Thermogravimetric Analysis of Residual Moisture Content

[0055] To determine residual moisture content of lyophilized LM products, the dehydrated samples are prepared in pre-weighed ampoules as described prior. Following dehydration, the sample ampoules are weighed and then heated at 110°C in an oven for 3 hours to evaporate the residual moisture. Following heating period, the sample ampoules are rapidly transferred to a vacuum desiccator and cooled to room temperature. The samples were then weighed again and residual moisture content calculated using the equation: ResidualMoisture=(SampleWeight- DrySampleWeight / SampleWeight)*100.Evaluation of Lyophilized Listeria In Vivo

[0056] For subcutaneous (s.c.) tumor studies comparing LM preparations 6-week old male Balb / c mice are implanted with Renca murine kidney cancer cells (n=8) as previously described. To briefly summarize, mice were implanted s.c. with 1x106Renca cells and treated beginning on day 5 when tumors became palpable. Treatments of LM LLO-CD105A antivascular vaccine were administered intraperitoneally with 2x108CFU suspended in 100 pL of PBS which were prepared identically to LM preparations described above. Tumors were measured every two days with perpendicular caliper measurements (V=lengthxwidth2 / 2) with treatments repeated every seven days following initial treatment.Statistical AnalysisPATENTAttorney Docket No.: TXTU / 0006PC

[0057] All statistical analysis was performed using GraphPad Prism 10 version 10.4.1. For comparison of lyophilized LM density, two-tailed unpaired student t-test was performed. Statistical analysis of tumor volume comparing control and stock / lyo- LM vaccine treatments was performed using one-tailed unpaired student f-test. Comparison of tumor immune infiltrates was performed using One-Way ANOVA with multiple comparison. Significant p values for all comparisons are illustrated in the following figures in the following way: ns p value > 0.05, *p value < 0.05.Examples and ResultsFreezer Stability of Lyophilized Listeria

[0058] The constituents (e.g., a lyoprotectant or a buffer component) in a lyophilization medium can impact bacterial viability. These bacteria can include a variety of LM serotypes, strains, and mutants which constituents can be effective at preserving. As shown in the Figures, the survival of the attenuated LM LLO-Ova vaccine strain was compared using established lyophilization mediums. The LM LLO- Ova strain was derived from the XFL7 LM strain transformed with pGG34 plasmid expressing truncated listeriolysin fused to ovalbumin and is similar to LM strains being investigated clinically.

[0059] Of the lyophilization media evaluated (shown in Table 1 ) using a colony forming unit (CFU) counting assay, three utilized a sucrose lyoprotectant (10% sucrose medium, phosphate sucrose medium, and American Type Culture Collection Reagent 18) while the fourth’s composition (Microbial Freeze-Drying Buffer from OPS Diagnostics) is unreported. As shown in Figure 2, following lyophilization, the 10% sucrose and phosphate sucrose media were found to have the highest LM survival. As shown in Figure 3, both the OPS Microbial Buffer and ATCC Reagent 18 were observed to cause significant bubbling during the lyophilization of LM as opposed to producing a dried powder like both 10% sucrose and phosphate sucrose media. Due to a higher LM viability and consistency when LM was prepared with the 10% sucrose and phosphate sucrose media, both were used for comparison in further LM lyophilization investigation.PATENTAttorney Docket No.: TXTU / 0006PC

[0060] Following initial investigation of LM lyophilization, stability was evaluated as a critical criterion of a viable lyophilization medium. Utilizing a CFU counting assay, both the 10% sucrose and phosphate sucrose media were observed to lose viability over a four-week period at -16°C to -20°C, as shown in Figure 1A. The 10% sucrose medium demonstrated the largest change in LM viability and the lowest consistency. Despite the reduced sucrose lyoprotectant content, the inclusion of a phosphate buffer improved the stability and consistency of the lyophilized LM. To determine whether the viability, stability, and consistency of lyophilized LM at -16°C to -20°C could be improved the application of alternative non-volatile buffers and sugar was investigated and disclosed herein.

[0061] To investigate alternative lyophilization buffers, three non-volatile buffers were selected: glutamate, 4-(2-hydroxyethyl)-1 -piper-azineethanesulfonic acid (HEPES), and triethanolamine (TEA). All three buffers have reported buffering ranges for maintaining a biological pH of 7.4. Glutamate was investigated because glutamate has reportedly demonstrated protective properties during lyophilization of lactic-acid bacteria. HEPES is unreported in the application of lyophilizing bacteria but is reported to be resistant to freezing-induced acidification. Additionally, HEPES has been demonstrated to mitigate the strong acidification associated with sodium phosphate buffers during freezing when used in combination and was investigated in combination with a phosphate buffer system. Finally, TEA is wholly unreported as a freezing or lyophilization buffer and was selected to investigate whether the only necessary criteria for a viable LM lyophilization buffer is to be non-volatile and have a buffer range around pH 7.4.

[0062] Figure 1A shows survival and consistency of LM LLO-Ova lyophilization media stored at -16°C to -20°C following preparation using various non-volatile buffers and sucrose as lyoprotectant (n = 3). LM were prepared for lyophilization by resuspension in a lyophilization medium, freezing at -80°C, lyophilization overnight, and sealing the ampoule before transferring to a freezer. The freezer is consistently maintained at a temperature of -16°C to -20°C. As shown in Figure 1A, evaluating the alternative buffers for lyophilized LM stability the HEPES sucrose medium was found to produce the highest viability and consistency of the buffers tested while maintaining a good stability over four-weeks at -16°C to -20°C. As shown in Figure 4,PATENTAttorney Docket No.: TXTU / 0006PC the stability was further confirmed to extend to three months at -16°C to -20°C with improved stability and consistency over the phosphate sucrose medium over the same period of time. When the phosphate and HEPES sucrose media were combined, however, the viability of lyophilized LM was significantly reduced (P=0.01 , One-way ANOVA) compared to HEPES alone. Lyophilization of LM using the glutamate sucrose medium resulted in fluctuating viability and lower overall consistency as compared to the phosphate and HEPES sucrose media. Similarly, the TEA sucrose medium resulted in fluctuating viability of the lyophilized LM, but a much more rapid reduction in survival and consistency as compared to both phosphate and HEPES sucrose media.

[0063] With HEPES buffer demonstrating the best protective effects of tested buffers, the effectiveness of trehalose as a lyoprotectant was also evaluated. Trehalose is another common lyoprotectant and so was investigated with the trehalose and HEPES trehalose media for LM stability at -16°C to -20°C. As shown in Figure 1A, lyophilization with both trehalose media resulted in growth of LM when stored at -16°C to -20°C. Both media also resulted in worse consistency than their sucrose counterparts demonstrating sucrose to be a superior lyoprotectant for the preparation and storage of LM. To further evaluate the phosphate and HEPES sucrose media when lyophilizing LM, the density of LM was investigated in the dehydrated powder products.

[0064] Following the powdered LM removal from ampoules, the powdered LM were weighed and then CFU counted to determine density of both dehydrated powders. Figure 1 B shows a comparison of CFU per milligram of phosphate and HEPES sucrose lyophilization media. Statistical comparison performed by two-tailed, unpaired T test (P =0.827; n = 3). As shown in Figure 1 B, both the phosphate sucrose and the HEPES sucrose media resulted in very similar densities of about 108CFU / mg of lyophilized LM. As shown in Figure 4, the long-term stability of the phosphate and HEPES sucrose medias with the LM over the course of three months were tested. While the phosphate sucrose media demonstrated a loss of consistency by three months, the HEPES sucrose media maintained stability and consistency over the same period. These results seem to indicate that the HEPES sucrose maintains the best stability of the LM vaccine over longer term storage at -16°C to -20°C.PATENTAttorney Docket No.: TXTU / 0006PCInfectivity of Lyophilized Listeria in J774 Cell Line

[0065] The process of lyophilization to dehydrate bacteria inherently results in some loss of cell viability. In the context of LM, a loss of viability potentially includes a loss of infectivity, ability to survive inside infected cells, and / or loss of utility as a therapeutic. To evaluate whether preparation of lyophilized LM impacts LM infectivity, a gentamicin protection assay (GPA) was used to compare LM LLO-Ova prepared with both phosphate and HEPES sucrose media at increasing multiplicities of infection (MOI). Figure 1 C shows the infectivity of LM in J774 cell line comparing standard stock LM stored at -80°C and derived phosphate and HEPES sucrose lyophilized LM (n = 1 agar plate CPU count per data point). LM infectivity post lyophilization was compared using a GPA wherein J774 cells were infected for 3 hours, washed, and then treated for 1 hour with 50 pg / mL gentamicin before cell lysis and CFU counting. As shown in Figure 1 C, both media were compared to the infectivity of nonlyophilized LM stored at -80°C in the macrophage-like sarcoma J774 cell line with singular CFU counts at multiple MOI. In the GPA, during which extracellular LM are killed with the cell impermeable antibiotic gentamicin, both the phosphate and HEPES sucrose medium demonstrated highly similar infectivity in J774. Both lyophilization media also resulted in a similartotal CFU to the non-lyophilized LM albeit still slightly lower (—0.1 -fold decrease in total CFU).

[0066] Figure 1 D shows the effects of LM lyophilization were secondarily evaluated using an SRB cytotoxicity assay wherein infected cells were washed after 3 hours of infection and incubated for 48 hours with 5 pg / mL gentamicin (n = 3). As shown in Figure 1 D, a GPA SRB cell viability assay was used to evaluate cytotoxicity in J774 to show the slight reduction in infectivity caused by lyophilizing LM impacts the LM life cycle. Following infection and incubation for 48 hours, both the phosphate and HEPES sucrose media demonstrated very similar cytotoxicity profiles to the nonlyophilized LM.Refrigeration Stability of Lyophilized Listeria

[0067] Storage of lyophilized LM at refrigeration (2°C to 4°C) temperatures is an inherently different environment compared to freezing temperatures. Bacteria grow more rapidly at 2°C to 4°C and, therefore, it is expected that lyophilized LM will havePATENTAttorney Docket No.: TXTU / 0006PC differing viability and stability at 2°C to 4°C as compared to -16°C to -20°C. Figure 1 E shows the survival and consistency of LM lyophilization media stored at 2°C to 4°C using sucrose and alternative lyoprotectants (n = 3). Following preparation lyophilized LM were transferred to refrigerator maintaining 4°C. As shown in Figure 1 E, when the 10% sucrose, phosphate sucrose, and HEPES sucrose media were used to prepare lyophilized LM LLO-Ova all three media demonstrated a lower one-day viability when stored at 2°C to 4°C. Both 10% sucrose and phosphate sucrose media demonstrated a fluctuating viability over a one-month storage period while the HEPES sucrose medium demonstrated a steady increase in viability over the same period. This increase in viability was likely caused by LM proliferation at 2°C to 4°C. During storage at -16°C to -20°C, it was observed that sucrose medium lost viability over time while trehalose increased in viability. The lyoprotectant sucrose is reportedly unable to be used as a carbon source by LM while trehalose can be used, indicating that non- metabolizable sugars may be more viable as lyoprotectants for bacteria preparation.

[0068] A variety of non-metabolizable lyoprotectants were tested for stability. Sucralose is a sucrose derivative artificial sweetener that has been postulated to have useful lyoprotectant properties. Sucralose, similar to sucrose, is taken up by a variety of microbes but has demonstrated antimicrobial activity due to increased reactive oxygen species generation and cell envelope damage. Since sucrose is not metabolized by LM, sucralose could be a uniquely viable lyoprotectant for LM. Xylitol is a sugar alcohol that has reportedly been used as a lyoprotectant and is unable to be metabolized by dental-plaque bacteria. Xylitol is also reportedly not a viable carbon source for LM. Cellulose is wholly unreported in conventional methods as a lyoprotectant and LM is reported to express the virulence factor Icp which facilitates cellulose binding. Cellulose is highly insoluble in water, which, in addition with Icp binding, could facilitate higher aggregation of LM around the cellulose lyoprotectant and promote improved survival.

[0069] As shown in Figure 3, the HEPES sucrose medium demonstrated the highest viability at -16°C to -20°C, because of this, the three non-metabolizable lyoprotectants were prepared with a HEPES buffer. Of the three media, as shown in Figure 3 the HEPES sucralose and HEPES cellulose media produced powdery products while the HEPES xylitol medium produced an amorphous product. As shownPATENTAttorney Docket No.: TXTU / 0006PC in Figure 1 E, the three alternative HEPES media demonstrated reduced viability at 2°C to 4°C as compared to the media using sucrose as a lyoprotectant. Of the three alternative lyopro tectan ts, sucralose demonstrated the lowest overall viability of LM indicating its cytotoxicity was not perturbed as a lyoprotectant for LM.SEM Imaging and Residual Moisture Content of Lyophilized Listeria

[0070] During investigation of the LM CFU density following lyophilization, it was demonstrated that both phosphate and HEPES sucrose media produce a high density of LM per milligram of dried product. While both media produce a high LM density, these measurements were not able to evaluate how well the LM is incorporated into the lyophilized powder. SEM imaging shows that the composition of a lyophilization medium can impact how well individual bacterium are incorporated into the powder product and, therefore, limit potential bacteria shedding during handling. Undesired shedding of LM from the powdered product poses a potential health concern to individuals handling the lyophilized LM product wherein there is a greater risk for surfaces to become contaminated. Therefore, both HEPES and phosphate media were prepared with and without LM and imaged using SEM to evaluate the impact of lyophilization on the dehydrated products. Figure 5A shows SEM images of HEPES sucrose lyophilization media and Figure 5B shows SEM images of phosphate sucrose lyophilization media. Figure 5C shows SEM images of HEPES sucrose lyophilization media with LM strain XFL7 and Figure 5D shows SEM images of phosphate sucrose lyophilization media with LM strain XFL7. The SEM images show an increase in lyophilized powder porosity and granularity with the inclusion of LM XFL7. Individual LM could not be clearly distinguished from the protectant matrix indicating LM are well incorporated into lyophilized powders.

[0071] As shown in Figure 5A and 5B, SEM imaging of the HEPES and phosphate media without LM revealed a high similarity in structure of the two media with a glasslike appearance. Detailed SEM analysis revealed a high uniformity in both media with minor deformations ranging approximately 5 pm to 20 pm in size. For the analysis of lyophilization media with LM, both the HEPES and the phosphate media were prepared with the LM strain XFL7. XFL7 is a highly attenuated LM strain with a prfA deletion and the strain used to derive LM LLO-Ova and other vaccine strains. AsPATENTAttorney Docket No.: TXTU / 0006PC shown in Figure 5C and 5D, SEM imaging of the lyophilized products including LM revealed a less uniform and more granular powder with distinct and scattered perforations. The perforations in both media varied in size and individual granules were notably smoother and less glasslike with the inclusion of LM. Neither the HEPES nor phosphate media when imaged in detail had visible LM on the surfaces of the powders indicating that the LM is well incorporated into the lyoprotectant.

[0072] Figure 6 shows the residual moisture content of lyophilized LM in both the phosphate and HEPES sucrose medias in comparison to the generic sucrose media. Thermogravimetric analysis was used to determine water content of lyophilized LM XFL7 and was chosen for its routine application with a wide variety of sample types from soil to bacteria. Both the Phosphate sucrose and HEPES sucrose medias were determined to produce products of similar residual moisture content levels (about 6.5% to about 7.5%) but higher than that of the sucrose media (about 5%). The residual moisture content was further found to match well with conventional values for overnight dehydration. Parameters such as residual moisture content potentially provide metrics for further improving on the methods described herein. Reduced residual moisture content as low as 1 % has been demonstrated to improve long-term stability over higher residual moisture content with other bacterial species. Further reducing the moisture content in the dehydrated LM would require increased drying time and could reduce initial viability, however, it could enable improved long-term storage (e.g., storage over three months) at -16°C to -20°C and 2°C to 4°C.In Vivo Comparison of Lyophilized Listeria

[0073] The viability of the lyophilized LM vaccine formulation to stimulate an immune response for targeted immunotherapy was explored in vivo. The application of an antivascular LM LLO-CD105A vaccine strain in the treatment of renal cell carcinoma (RCC) was investigated. CD105 is commonly associated with tumor- associated vasculature but, in RCC, it also promotes immunosuppression, metastasis, and reduced survival. The LM LLO-CD105A vaccine strain was constructed to express a subunit of the CD105 protein which, when applied in RCC, was demonstrated to significantly reduce tumor growth in a subcutaneous (s.c.)PATENTAttorney Docket No.: TXTU / 0006PCRenca cell model. This efficacy was demonstrated to promote immune stimulation and is CD8+ T cell dependent.

[0074] Figure 7 A shows a schematic illustration of subcutaneous Renca renal cell carcinoma (RCC) implantation and intraperitoneal (i.p.) treatment. 1x106Renca cells were implanted into male Balb / cJ mice and tumor growth kinetics monitored with treatment beginning five days post-implantation. As shown in Figure 7A, to evaluate and compare the efficacy of lyophilized LM in vivo, Renca cells were implanted s.c. in mice and treated with non-lyophilized and lyophilized preparations of LM LLO- CD105A. Tumors became palpable on the day of first treatment, five days post implantation.

[0075] Non-lyophilized LM LLO-CD105A treatments were prepared from standard -80°C stocks while the lyophilized LM was prepared with the HEPES sucrose medium and stored at -16°C to -20°C. Figure 7B shows tumor growth kinetics of mice treated with PBS (control), non-lyophilized stock LM LLO-CD105A, and lyophilized LM LLO- CD105A (mean ± SEM). Statistical comparison performed by one-tailed unpaired T- test (*P < 0.05; n = 8). Figure 7C shows individual tumor growth kinetics for PBS, non-lyophilized LM, and lyophilized LM. As shown in Figures 7B and 7C, both treatments significantly reduced tumor growth compared to control treatment with both non-lyophilized and lyophilized treatments resulting in very similar mean tumor volumes. Figure 8A and Figure 8B shows immune infiltration of (8A) leukocytes (CD45+) and (8B) MDSC (CD11 b+ GR1 hi) in RENCA tumor cell environment post treatment with PBS, non-lyophilized stock LM LLO-CD105A, and lyophilized LM LLO- CD105A analyzed by flow cytometry. Statistical comparison performed by One-Way ANOVA (*P<0.05; n=5).

[0076] As shown in Figures 8A, unexpectedly, despite similar efficacy in the s.c. Renca tumor model, when analyzed for immune infiltration, it was observed that the lyophilized LM LLO-CD105A treated tumors had a larger leukocyte (CD45+) population despite treatment with the same amount of viable LM. As shown in Figure 8B, the increased immune infiltration in the lyophilized LM treated tumors further corresponded to an increased myeloid derived suppressor cell (MDSC; CD11 b+GR1hi) population. The in vivo comparisons of lyophilized LM LLO-CD105A vaccinePATENTAttorney Docket No.: TXTU / 0006PC treatment indicate that overall efficacy is not affected but differences in immune recruitment could enhance therapeutic combinations. The lyophilized LM LLO- CD105A vaccine inhibited tumor growth equivalently to the non- lyophilized vaccine in a subcutaneous Renca kidney cancer model. While the lyophilized LM LLO- CD105A treatment resulted in equivalent efficacy, there was also an observed increase in leukocyte (CD45+) and MDSC (CD11 b+GR1hi) infiltration in the lyophilized LM LLO-CD105A treatment.Encapsulation

[0077] As shown in Figure 9, SRB release from coated M size capsules under (A) simulated intestinal fluid and (B and C) simulated gastric fluid overtime. SRB capsule payloads are released rapidly under intestinal conditions with methacrylic acid copolymer (e.g., Eudragit S-100) and Polycaprolactone (PCL) prolonging release under gastric conditions thereby enabling intestinal targeted delivery.

[0078] As shown in Figure 10, visualization of M size capsules uncoated and coated with (A) methacrylic acid copolymer (e.g., Eudragit S-100) and (B) PCL release of SRB dye under simulated gastric and intestinal conditions. Capsule coating delays capsule payload release under gastric conditions enabling controlled release in the intestines improving payload stability and targeting.

[0079] As shown in Figure 11 , in vivo treatment of orthotopic mammary fat pad implanted EO771 breast cancer with LM LLO-CD105A antivascular vaccine strain. Mice were either untreated, treated with orally administered LM vaccine, or treated with encapsulated LM (1-2 capsule doses) coated with a methacrylic acid copolymer (e.g., Eudragit S-100) to enable lower intestine targeting. Capsule delivery to the lower intestine enabled by a methacrylic acid copolymer (e.g., Eudragit S-100) coating reduced tumor growth at distal tissue site indicating successful induction of anticancer mucosal immunity.

[0080] Together, the disclosed data demonstrates the preparation of lyophilized LM in a powder form is compatible with encapsulation for targeted delivery to mucosal sites, such that the lyophilized powder is operable to be a mucosal vaccine platform. This approach of encapsulation is a significant improvement to typical LMPATENTAttorney Docket No.: TXTU / 0006PC preparations for targeting LM vaccine delivery to mucosal sites by directly delivering LM to pro-inflammatory and, therefore, anti-tumoral mucosal tissue regions. Previous LM dosing approaches promote systemic immune responses poorly suited to targeting mucosal diseases or result in poor survival of administered LM and fail to produce a robust pro-inflammatory mucosal immunity.

[0081] The disclosed lyophilization method (method 1200) and encapsulation and coating protocol facilitate storage of a LM based therapeutic at cold chain viable temperatures. LM is operable to be freeze dried while maintaining viability. The choice of lyoprotectant and buffer components in the lyophilization medium greatly impacts the viability and stability of the lyophilized LM. Similarly, the choice of coating during the encapsulation process impacts the delivery of the therapeutic within a subject.

[0082] Since preparation with sucrose results in loss of LM viability over time while trehalose results in an increase in viability over time when stored at -16°C to -20°C, it is possible a “sweet spot” exists with a mixture of lyoprotectants that confers greatly improved stability. From a therapeutic dosing perspective, it is preferred that a stock lose effective concentration over time rather than increase so as to prevent accidental overdosing of subjects and, therefore, should be handled carefully.

[0083] Overall, embodiments of the present disclosure disclose a lyophilization media for the preparation and storage of LM anti-cancer vaccine strains under frozen and refrigerated conditions. Firstly, recognizing the current challenges of ULT distribution of LM vaccines and lack of conventional methods on LM lyophilization. Accordingly, methods of LM dehydration by lyophilization that maintained LM viability were pursued. A variety of lyophilization media for overall viability and survival was evaluated at -16°C to -20°C and 2°C to 4°C. Of the lyophilization media tested, the 3 mM HEPES 2.5 % w / v sucrose medium was found to have the best viability, stability, and consistency at -16°C to -20°C during four-week storage comparison and extended stability to at least three months. The stability of lyophilized LM at -16°C to -20°C was found to not extend to the 2°C to 4°C storage condition. The lyophilized LM was further observed to be well-incorporated within the lyophilization medium and in in vivo comparison was unperturbed in efficacy. The methods and formulationsPATENTAttorney Docket No.: TXTU / 0006PC presented herein are expected to significantly improve the availability and ease of storage of LM anti-cancer vaccines and potentially other LM therapies. Further, an encapsulation and coating method is disclosed. After lyophilization of the LM, the lyophilized LM can be further processed by encapsulation for easy delivery to a subject.

[0084] While the experimental methodology and results include mouse subjects, the subject matter disclosed herein is applicable and for human use. The terms “subject”, “individual” or “patient” are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, mice and humans. The lyophilized powder may be administered to a nonhuman mammal for the purposes of obtaining preclinical data, for example. Exemplary nonhuman mammals to be treated may include nonhuman primates, dogs, cats, rodents (such as rats or mice), or other mammals in which preclinical studies are performed. Such mammals may be established animal models for a disease to be treated with the lyophilized powder or may be used to study toxicity of the lyophilized powder of interest.

[0085] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. I n aspects, use of the term “about” may refer to ±20% of the stated value, ±15% of the stated value, ±10% of the stated value, ±5% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1 % of the stated value.

[0086] As used herein, a “composition” may include component(s) of the composition, reaction product(s) of two or more components of the composition, a remainder balance of remaining starting component(s), or combinations thereof. Embodiments described herein also related to LM based therapeutics for treating medical conditions such as cancer.PATENTAttorney Docket No.: TXTU / 0006PC

[0087] “Therapeutics,” “treatment,” and “treating” include the medical management of a subject with the intent to cure, ameliorate, or stabilize a disease, pathological condition or disorder (e.g., cancer). This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment that is directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

[0088] While the foregoing is directed to aspects of the present disclosure, other and further aspects of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

PATENTAttorney Docket No.: TXTU / 0006PCWhat is claimed is:

1. A lyophilized powder, comprising: a Listeria monocytogenes (LM) bacteria; and a lyophilization medium comprising: a lyoprotectant; and a buffer component.

2. The lyophilized powder of claim 1 , wherein the lyoprotectant comprises sucrose.

3. The lyophilized powder of claim 1 , wherein the buffer component comprises 4- (2-hyrdoxyethyl)-1 -piperazineethanesulfonic acid (HEPES).

4. The lyophilized powder of claim 1 , wherein the LM bacteria comprises at least one therapeutic agent.

5. The lyophilized powder of claim 1 , wherein the lyophilization medium comprises about 1 millimolar (mM) to about 10 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and about 2% to about 10% weight / volume (w / v) sucrose.

6. The lyophilized powder of claim 1 , wherein the lyophilized powder maintains a percent survival of about 50 percent to about 100 percent at a temperature of about - 16°C to about -20°C.

7. The lyophilized powder of claim 1 , wherein the LM bacteria further includes a therapeutic agent, wherein the therapeutic agent comprises a saporin, an active metabolite of CPT-11 , a doxorubicin, derivatives thereof, or combinations thereof.

8. A Listeria monocytogenes (LM) based vaccine formulation, comprising: a lyophilized powder comprising:PATENTAttorney Docket No.: TXTU / 0006PCLM bacteria, the LM bacteria comprising at least one therapeutic agent; and a lyophilization medium; and a delivery composition.

9. The LM based vaccine formulation of claim 8, wherein the lyophilization medium comprises a lyoprotectant and a buffer component.

10. The LM based vaccine formulation of claim 9, wherein the buffer component comprises 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES).11 . The LM based vaccine formulation of claim 9, wherein the lyoprotectant is sucrose.

12. The LM based vaccine formulation of claim 11 , wherein the lyophilization medium comprises about 1 mM to about 10 mM 4-(2 hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) and about 2% to about 10% w / v sucrose.

13. The LM based vaccine formulation of claim 8, wherein the delivery composition is a capsule operable to hold the lyophilized powder.

14. The LM based vaccine formulation of claim 13, wherein the capsule comprises a coating comprising methacrylic acid copolymer, polycaprolactone (PCL), cellulose derivatives, polyvinyl derivatives, or combinations thereof.

15. The LM based vaccine formulation of claim 8, wherein the delivery composition is a liquid for resuspension, the liquid for resuspension operable to resuspend the lyophilized powder.

16. A method of forming a Listeria monocytogenes (LM) based vaccine formulation, comprising:PATENTAttorney Docket No.: TXTU / 0006PC resuspending an LM stock in a lyophilization medium to form a lyophilization composition, wherein the lyophilization medium comprises at least a lyoprotectant and a buffer component; freezing the lyophilization composition; lyophilizing lyophilization composition; and forming a lyophilized powder, the lyophilized powder comprising: a LM bacteria; the lyoprotectant; and the buffer component.

17. The method of claim 16, wherein the lyophilization medium is about 1 mM to about 10 mM 4-(2-hyrdoxyethyl)-1 -piperazineethanesulfonic acid (HEPES) and about 2% to about 10% w / v sucrose.

18. The method of claim 17, wherein lyophilizing the lyophilization composition further comprises: exposing the lyophilization composition to a vacuum pressure; and exposing the lyophilization composition at a temperature of about -30°C to about -80°C; and wherein the lyophilization composition is exposed to the vacuum pressure and the temperature until the lyophilized powder is formed.

19. The method of claim 17, further comprising: encapsulating the lyophilized powder within a capsule; and coating the capsule with a methacrylic acid copolymer, polycaprolactone (PCL), cellulose derivatives, polyvinyl derivatives, or combinations thereof.

20. The method of claim 19, wherein the capsule is operable to encapsulate about 100 million colony forming units (CPU) to about 300 million CPU of the LM bacteria.

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